1. Field of the Invention
The present invention relates to a diagnostic assay for mycoplasma infections. The assay is based on antigen-antibody reaction and is effective in differentiating specific mycoplasma infection by species.
The ability to identify specific species of mycoplasma infection can be of great value. In addition to improving specific diagnosis, prognostication, knowledge of the particular mycoplasma species causing the infection allows the attending physician to select the most appropriate therapy for the individual patient, e.g. highly aggressive or less aggressive therapy regimens. Different species of mycoplasma often show different sensitivity to various antibiotics. Because of patient distress caused by more aggressive therapy regimens, it is desirable to distinguish those patients requiring such therapies.
Mycoplasma is a genus of cell wall-less sterol-requiring, catalase-negative pathogens commonly found in the respiratory and urogenital tracts of man and other animals. The cells of Mycoplasma are typically non-motile and pleomorphic, ranging from spherical, ovoid or pear-shaped to branched filamentous forms. Filaments are the typical forms in young cultures under optimal conditions, which subsequently transform into chains of coccoid cells which later break up into individual cells that are capable of passing through membrane filters of pore size 0.45 .mu.m or even 0.22 .mu.m.
A trilaminar cytoplasmic membrane contains sterols, phospholipid and proteins. Therefore, the cells are generally susceptible to polyene antibiotics and to lysis by digitonin.
Replication of the Mycoplasma genome may precede cytoplasmic division resulting in multinucleate filaments before individual cells are delimited by constriction. Budding can also occur.
Most Mycoplasma species are facultatively anaerobic, and all known species are chemoorganotrophic. The fermentative species of Mycoplasma utilize sugars such as glucose, while non-fermentative species can hydrolyze arginine. Only a few species of mycoplasma can utilize both glucose and arginine to grow.
Known mycoplasmas may be grown on complex media, such as Hayflick medium, while fastidious mycoplasmas may be grown on diphasic SP-4 medium. The colonies are usually of the "fried egg" type, i.e., an opaque, granular central region, embedded in the agar, surrounded by non-granular surface growth. The optimal growth temperature of mammalian strains is 36.degree.-37.degree. C.
Many species of Mycoplasma produce weak or clear hemolysis which appears to be due to the secretion of H.sub.2 O.sub.2. This H.sub.2 O.sub.2 secretion is believed to be responsible for some aspects of the mycoplasmas' pathogenicity. Known mycoplasmas are commonly sensitive to chloramphenicol and tetracyclines.
The Mycoplasma genus currently consists of more than 60 known species which are differentiated on the basis of various tests, including utilization of glucose and mannose, arginine hydrolysis, phosphatase production, the "film and spots" reaction and haemadsorption.
Mycoplasmas are the smallest and simplest free-living organisms known. Mycoplasmas are not obligatory intracellular microorganisms and are usually found extracellularly, but can be found intracellularly in the infected tissues (Mycoplasma, Eds. Wolfgang, J. J., Willette, H. P., Amos, D. B., Wilfert, C. M., Zinsser Microbiology 19th Ed. 1988, Appleton and Lange, 617-623). The term mycoplasma apparently was first used by B. Frank in 1889 (Frank B., Dent. Bot. Ges., 7, 332 (1889) and Krass, C. J. et al., Int. J. Syst. Bacteriol. 23, 62 (1973)). Frank, after careful microscopic observation, began writing about invasion of plants (legume) by these microorganisms and stated: "the changed character of the protoplasm in the cortical cells arising from infection, I will designate as mycoplasma". Later, he had more explicitly defined mycoplasma as a mixture of small fungus-like microorganisms and cell protoplasm (Frank, B., Landwirt. Jahrb. 19, 523 (1890)). The description reflected the difficulty of differentiating this unique microorganism from the infected host cells morphologically.
Even today with electron microscopy, it is still often difficult to differentiate the mycoplasmas from the cellular protoplasmic processes or the subcellular organelles of the infected host, because ultrastructurally, these have microorganisms have protoplasm-like internal structures and are bounded by only an outer limited membrane (unit membrane) without a cell wall. Thus, there have been few electron microscopic studies of mycoplasmas identified directly in the infected tissues of animals or humans.
It has been reported that ultrastructural examination of infected tissues has failed to localize the microbe, even in tissues where very high titers (&gt;10.sup.9 /gm) of microorganisms were recovered in culture (Elizan, T. S. et al., Pro. Soc. Exp. Biol. Med. 139, 52 (1972) and Schwartz, J. et al., Pro. Soc. Exp. Biol. Med. 139, 56 (1972)). Therefore, morphologically, the microbe might be mimicking certain normal cellular or subcellular structures in the infected host tissues and preventing direct identification.
In addition to the natural difficulty of morphological differentiation between the microorganisms and the protoplasm of infected cells, the often poorly preserved formalin-fixed clinical materials present further limitations to any attempt to directly visualize mycoplasma organisms in the tissues.
Mycoplasmas cause a variety of diseases in animals, plants and insects. However, the roles of known human mycoplasmas except for M. pneumoniae in causing atypical pneumonia are difficult to assess. Urines or urogenital swabs from healthy people and patients with a variety of different diseases have already been examined extensively. U. urealyticum and M. hominis were the most commonly found mycoplasmas. Taylor-Robinson, D., et al., Nature 222, 274 (1969); Tarr, P. I., et al., J. Infec. Dis 133, 419 (1976); Taylor-Robinson, D., et al., N. Eng. J. Med. 302, 1003 (1980); Fiacco, V., et al., J. Clin. Microbiol. 20, 862 (1984). Although a wide variety of urogenital and reproductive disorders have been associated with U. urealyticum and M. hominis, it has been difficult to establish a definite etiological role for them because they are often found as frequently in patients without disease as in those with disease. Taylor-Robinson, D., et al., N. Eng. J. Med. 302, 1003 (1980). M. genitalium was also isolated from two patients with non-gonococcal urethritis. However, most patients with urogenital diseases do not have clear evidence of M. genitalium infection. Tully, J. G., et al., Lancet 1, 1288 (1981). On the other hand, mycoplasmas are known to be extraordinary pathogens, capable of causing chronic debilitating diseases and producing a variety of clinical manifestations in animals and frequently suppressing host immune defense mechanisms. The Mycoplasmas, Vol. IV, Razin, S., Barile, M. F., eds., Academic Press, pp. 203-286 (1983).
2. Description of the Background Art
As noted above, mycoplasmas are currently differentiated by various tests for their unique biological and biochemical properties including utilization of glucose and mannose, arginine hydrolysis, phosphatase production, the "film and spots" reaction and haemadsorption. Classical serologic tests for detection of antibodies to either whole organisms or purified fractions include: indirect fluorescent antibodies (whole organisms), complement fixation, passive hemagglutination, agglutination, growth inhibition on agar and metabolic inhibition in broth medium. Also, an immunochemical assay for Mycoplasma genitalium using specific monoclonal antibodies in sandwich ELISA binding assay has been claimed in U.S. Pat. No. 5,158,870. However, a sensitive and specific assay detecting antibodies to each specific species of mycoplasma is still urgently needed in the clinics.
At least part of the reason for the lack of such an immunological assay is the significant cross reactions of mycoplasma proteins between different species of mycoplasma, as well as the reported tendency of mycoplasmas to rapidly alter expression and structure of their lipid-modified, cell-surface proteins. Rosengarten, R. and Wise, K., Science 247, 315 (1990). In fact, differences in the expression (phase variation) and in size of lipid-modified, integral membrane protein antigens at the cell surface have been reported among progenies from a single mycoplasma organism, and render development of a serological assay to the ever changing lipid-modified integral membrane protein antigen unlikely. Id. Thus, an accurate immunological diagnostic assay to detect mycoplasma infections and to specifically distinguish between different species of mycoplasma would represent a significant advancement in the art.